Project Summary: Muscle ciliary neurotrophic factor receptor ? (CNTFR?) expression is induced: 1) by denervating nerve lesion53-55, 2) in human denervating diseases56,57, including ALS56, and 3) in all ALS models tested. Muscle CNTFR? knockdown inhibits motor neuron (MN) axon regeneration and motor recovery after nerve lesion53 and accelerates disease in all the ALS models, suggesting the muscle CNTFR? induction is a neuroprotective response that could be enhanced to treat ALS. We increased muscle CNTFR? in SOD1G93A ALS mice with an AAV vector (AAV1.1-CNTFR?), extending survival and increasing motor function (without side effect) even when started well after symptom onset, making it arguably the most clinically promising treatment to date since human ALS is not treated until well after symptom onset58-61. We find vector-derived CNTFR? protein translocated to MNs and increased MN terminals, suggesting a mechanism of action. This treatment should: 1) inhibit most/all ALS, 2) work independent of ALS causes, and 3) be translatable since muscle expression can be increased with approved human gene therapy techniques42-47,49,50, like those we use here. The ligand most likely involved in muscle CNTFR?'s anti-ALS effects is muscle cardiotrophin-like cytokine (CLC). We similarly increased muscle CLC in SOD1G93A mice, again extending survival without side effect, making it another very promising new ALS treatment. Combining the two treatments suggests this could be an even more effective treatment. We will: Aim 1: Optimize and Characterize muscle CNTFR? enhancement as an ALS treatment. To maximize effect, we will dose response test in SOD1G93A mice: 1) a codon optimized CNTFR? cDNA, 2) an AAV capsid with greatly enhanced muscle transduction (AAV1.1), 3) a muscle specific promotor (tMCK), 4) self-complementary AAV for faster and greater expression, and 5) IV injection of another next-gen capsid (AAV2i8) for transduction of more muscles. The best treatment will be further examined with: 1) rotarod and grip strength tests, 2) qRT-PCR and in situ hybridization for vector- derived CNTFR? RNA, 3) HA tag anatomy to localize vector-derived CNTFR? protein and identify potential sites of action, 4) multi-label immunohistochemistry to determine effects on ALS degeneration, and 5) two other diverse ALS models (SOD1G37R and TDP-43Q331K mice) to broadly test the treatment. Aim 2: Optimize and Characterize muscle CLC enhancement as an ALS treatment. We will run experiments exactly as in Aim 1, except with CLC instead of CNTFR?. Aim 3: Optimize and Characterize combined CNTFR? and CLC treatment. Muscle CLC and CNTFR? are likely released as a MN protective CLC/CNTFR? complex such that an increase in both CLC and CNTFR? may further enhance efficacy. A pilot with a 1:1 ratio of the non-optimized CLC and CNTFR? vectors found a substantially enhanced effect in females. We will test other ratios with CNTFR? and CLC treatments optimized in Aims 1 and 2, to further increase the female effect and potentially enhance effect in males. Best treatments will then be fully characterized as in Aims 1 and 2.